PROJECT SUMMARY/ABSTRACT Title: Novel Role of Myeloid-derived Lymphatic Progenitors in Induction of Breast Cancer Lymphatics Metastasis to lymph nodes, a common occurrence in breast cancer (BC), is the most significant prognostic indicator of poor outcome. To reach locoregional lymph nodes, tumor cells exclusively use lymphatic vessels. Not surprisingly, the extent of lymphatic metastasis is directly proportional to the density of tumor lymphatic vessels. It is therefore of high clinical significance to understand the mechanisms of tumor induced lymphangiogenesis, that is, the formation of new lymphatic vessels. It is currently thought that the main mechanism causing the formation of new lymphatic vessels is mediated by a paracrine lymphangiogenic factor VEGF-C that activates its receptor VEGFR-3 expressed in lymphatic endothelial cells. We recently discovered a fundamentally different mechanism of tumor lymphangiogenesis that complements the current views. This mechanism is mediated by tumor-mobilized bone marrow (BM)- derived monocytic progenitors that upon influence of the inflammatory tumor environment differentiate into lymphatic-like cells. These cells dubbed here Monocyte-derived Lymphatic Endothelial Cells Progenitors or M- LECP are characterized by the two main traits: (1) co-expression of myeloid and lymphatic-specific proteins that are typically segregated into distinct lineages; and (2) the ability to integrate into preexisting lymphatic vessels, which is an early prerequisite for lymphatic outgrowth. Using these criteria, we found very high levels of M-LECP in blood and tumors of BC patients as well as in a variety of metastatic orthotopic breast tumors from human and mouse origins. All tumors that contained M- LECP also displayed lymphatic vessels positive for myeloid-specific markers, an established phenomenon indicative of vascular integration of M-LECP that is required for sprouting. Importantly, we recently established that the levels of tumor-recruited M-LECP significantly correlate with tumor lymphatic density and lymph node status in clinical BC patients. Our studies in BC models showed that M-LECP originate from BM-derived CD11b+ cells that are highly positive for a lymphatic marker Podoplanin (Pdpn). Adoptive transfer of a phenotypically distinct BM subset identified by CD11b and Pdpn from metastatic tumor-bearing mice to mice with low-metastatic tumors significantly increased the density of lymphatic vessels and lymphatic metastasis. Preliminary data also show that differentiation of M-LECP can be faithfully reproduced in vitro by activating Toll-like Receptor-4 (TLR4) in human primary normal blood-circulating monocytes. This process is controlled by NF-kB and a transcription factor c-Maf, a newly identified regulator of monocytic-lymphatic reprogramming. TLR4-dependent upregulation of NF-kB and c-Maf leads to activation of the VEGFR-3 pathway which appears to be a critical milestone for acquisition of the lymphatic phenotype. Based on these findings, we hypothesize that tumor lymphatic outgrowth, an essential prerequisite for metastasis, is strongly promoted by M- LECP differentiating from the bone marrow myeloid precursors by TLR4-activating factors. To test this hypothesis we propose the following Specific Aims: (1) Test the hypothesis that M-LECP are directly and significantly relate to tumor-induced lymphangiogenesis and lymphatic metastasis in clinical BC; (2) Delineate the molecular mechanisms that reprogram bone marrow myeloid precursors into M-LECP; and (3) Determine the role of TLR4 in generation of the CD11b+/Pdpn+ bone marrow subset and its direct contribution to the formation of tumor lymphatic vessels and metastasis. Impact and Translational Relevance: We anticipate that these studies will establish a novel mechanism by which BM-derived monocytes recruited by breast tumors promote lymphangiogenesis and metastasis to lymph nodes. Validation of this novel concept will enhance the mechanistic understanding of the formation of tumor lymphatic vessels and suggest new molecular targets for inhibiting differentiation of M-LECP. Additionally, detection of higher levels of tumor- residing and blood-circulating M-LECP might identify BC patients with more aggressive tumors who should be preemptively treated to suppress locoregional spread. Such study outcomes can bolster the current clinical paradigms thus reducing mortality of BC patients.